A major limitation in determining the roles in pathogenesis of bacterial proteins is the difficulty of genetically manipulating mammalian cells. This grant proposes novel and efficient genome-wide screens with the genetically tractable yeast Saccharomyces cerevisiae to identify eukaryotic cellular pathways targeted by pathogenic Shigella proteins. In previous work the Principal Investigator has demonstrated that interactions between host cells and virulence proteins of Yersinia and Salmonella are conserved among yeast and mammals. This is not surprising given the high degree of conservation of the basic cellular processes among all eukaryotes. Remarkably, when expressed in yeast, 7 of the 18 proteins recently proposed to be delivered by the Shigella type III secretion system directly into mammalian cells confer a toxic yeast phenotype and/or localize to a specific subcellular compartment. Specific Aim 1 will compare the subcellular localization patterns of the Shigella proteins observed in yeast and mammalian cells. Specific Aim 2 describes genome-wide semi-automated phenotypic screens to identify eukaryotic proteins whose overexpression or loss of expression suppresses or exacerbates the toxic phenotypes conferred by individual pathogenic Shigella proteins. Computational analyses of the results of these complementary screens will generate systems-level insights into the eukaryotic cellular pathways targeted by individual Shigella proteins. Although these approaches are exploratory in nature, preliminary studies suggest that they are likely to generate hypotheses as to the targets of specific pathogenic proteins to test in more complex physiologic models of disease. While these studies focus on probable Shigella virulence proteins, these analyses are applicable to any microbial pathogen that targets eukaryotic intracellular processes. Thus, approaches developed in this grant to establish yeast as a model system for genome-wide analyses to determine the targets of pathogenic proteins, should prove particularly useful for organisms that are difficult to genetically manipulate or dangerous to grow.